CN1340549A - Process for preparing glucagon - Google Patents
Process for preparing glucagon Download PDFInfo
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- CN1340549A CN1340549A CN 00112488 CN00112488A CN1340549A CN 1340549 A CN1340549 A CN 1340549A CN 00112488 CN00112488 CN 00112488 CN 00112488 A CN00112488 A CN 00112488A CN 1340549 A CN1340549 A CN 1340549A
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- hyperglycemic
- glycogenolytic factor
- column
- precipitation
- exchange
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Abstract
A process for preparing glucagon uses the waste mother liquid of insuline sodium salt crystal as raw material, and includes such steps as Zn deposition or diluting the mother liquid, regulating pH value, and feeding into cation-exchange gel column to prepare glucagon.
Description
The invention belongs to the preparation method of biochemical drug hyperglycemic-glycogenolytic factor.
Hyperglycemic-glycogenolytic factor is present in a one cell of pancreas Lan Shi island, the polypeptide hormone of being made up of two nineteen amino-acid residues.Because separation and purification is difficulty, hyperglycemic-glycogenolytic factor runs off with mother liquor in the process of producing Regular Insulin.At present majority state all can not be in insulin production technology the separation and purification hyperglycemic-glycogenolytic factor, Tianjin second biochemical-pharmaceutical factory and biology department of Nankai University cooperating research from the method for animal pancreas separation and purification hyperglycemic-glycogenolytic factor, still be in testing laboratory's stage at present, its separation and purification flow process is:
Directly extract but this technology still belongs to from pancreas, the insulin crystals mother liquor still can not obtain utilizing.
The purpose of this invention is to provide a kind of technology that from depleted insulin sodium salt crystalline mother solution, prepares hyperglycemic-glycogenolytic factor, turn waste into wealth, improve overall economic efficiency.
Technical scheme of the present invention is as follows:
Waste insulin sodium salt crystalline mother solution with production Regular Insulin is a raw material, mother liquor is transferred PH3-5 with 1-3N hydrochloric acid, with centrifugal or remove by filter the acidic protein of separating out, the zinc acetate or the zinc chloride that in filtrate, add 0.01-0.1M then, the PH5-7.5 precipitation is transferred with 1-3N sodium hydroxide in the back, to precipitate with dilute acetic acid or diluted hydrochloric acid dissolution Shangyang ion-exchange gel post, use the weak ammonia wash-out then, elutriant will be added Zn again
++Secondary sedimentation, the Zn precipitation is dissolved with the saliniferous dilute acetic acid, last gel-filtration column, the hyperglycemic-glycogenolytic factor honeybee that gel-filtration column is washed out concentrates through cationic exchange coloum, then concentrated solution is transferred the PH7-9 crystallization with dilute phosphoric acid, use the phosphoric acid buffer systems recrystallization again, crystallization gets 12 body hyperglycemic-glycogenolytic factor crystal powders after collection, dehydration, drying, and its technical process is:
Also 2-10 times of volume of insulin sodium salt crystalline mother solution thin up can be replaced Zn with going up cationic exchange expanding bed post behind the 1-3N hydrochloric acid accent PH3-5 again in the aforesaid method
++Precipitation.
Embodiment one:
The insulin sodium salt crystalline mother solution transfers PH3-5 to remove by filter acidic protein with 1-3N hydrochloric acid, adds 0.01M zinc acetate (or zinc chloride) in the filtrate.3N sodium hydroxide is transferred PH4, leaves standstill, treat that precipitation fully after, centrifugal collections Zn precipitates.The concentration that the Zn precipitation is dissolved into about 5% with dilute acetic acid removes by filter insolubles, the cationic displacement chromatography post of last Sp sepharose XL.The post bed is used 0.2M acetic acid balance in advance.After the exchange of sample liquid upper prop, when having hyperglycemic-glycogenolytic factor to pass, stop exchange with HPLC detection effluent liquid.The post bed changes and is washed to PH6 with an amount of 0.2M dilute acetic acid balance.Change 0.2M ammoniacal liquor wash-out, elution peak has only one, presses aforesaid Zn after the collection again
++The precipitator method are carried out secondary Zn precipitation.After treating that precipitation is fully, centrifugal collection zinc precipitation.Get zinc precipitation 50 grams, use 0.2M acetic acid--the 0.1M sodium chloride solution removes by filter insolubles with the volume dissolving of minimum.Sephacryl S-100HR gel-filtration column (post bed 10 * 90cm) on the solution.The same lysate of control line flow velocity 23cm/h elutriant, the hyperglycemic-glycogenolytic factor peak that washes out (HPLC detection) concentrates through sp SepharoseXL post bed, elutes from the post bed with 0.2M ammoniacal liquor.Elutriant is transferred PH8.5 with the 6N dilute phosphoric acid, and put in the cold house, 5 ℃ of left and right sides and place crystallization, after 2-3 days, centrifugal collection crystallization.The 0.01M dissolving with hydrochloric acid is used in crystallization again, adds 2% urea, adds the PH6.8 phosphoric acid buffer of 1/10 volume, and heating removes by filter insolubles about 50C, when treating that solution temperature is reduced to 20 ℃ of room temperatures, adds 10% acetone.Proofread and correct PH6.8, recrystallization in the amount cold house, 5 ℃ of left and right sides, crystallization is 12 bodies.Centrifugal collection crystallization after 3 days, crystallization be through cold water washing, and cold ethanol is anhydrous, the anhydrous dehydration of cold diethyl ether, drying.Promptly obtain the hyperglycemic-glycogenolytic factor crystal powder, HPLC purity is again more than 95%.
Embodiment two: the insulin sodium salt crystalline mother solution, and 8 times of volumes of thin up are transferred PH4 with 3N hydrochloric acid, sp Sepharose XL cationic exchange expansion column on the solution, the post bed is used 0.2M acetic acid balance in advance.After the exchange of sample liquid upper prop, detect effluent liquid, when having hyperglycemic-glycogenolytic factor to pass, stop exchange with PHLC.The post bed is with an amount of 0.2M acetum balance.Change again and be washed to PH6, change 0.2M ammoniacal liquor wash-out, collect the back and carry out the Zn precipitation by the Zn precipitator method, treat that precipitation fully after, centrifugal collection zinc precipitation.Get zinc precipitation 50 grams, use 0.2M acetic acid--the 0.1M sodium chloride solution removes by filter insolubles with the volume dissolving of minimum.Sephacryl S-100HR gel-filtration column (post bed 10 * 90cm) on the solution.Control line flow velocity 23cm/h, the hyperglycemic-glycogenolytic factor peak that elutriant washes out with lysate (HPLC detection) concentrates through sp Sepharose XL post bed.Elute from the post bed with 0.2M ammoniacal liquor, elutriant is washed phosphoric acid with 6N and is transferred PH8.5, puts in the cold house, 5 ℃ of left and right sides and places crystallization, centrifugal collection crystallization after 3 days.The 0.01M dissolving with hydrochloric acid is used in crystallization again, adds 2% urea, adds the PH6.8 phosphoric acid buffer of 1/10 volume, heat and remove by filter insolubles about 50 ℃, when treating that solution temperature is reduced to 20 ℃ of left and right sides, add 10% acetone, proofread and correct PH6.8, put recrystallization in the cold house, 5 ℃ of left and right sides, crystallization is 12 bodies, centrifugal collection crystallization after 3 days, and crystallization is through cold water washing, cold ethanol (anhydrous), cold diethyl ether (anhydrous) dehydration, drying promptly obtain the hyperglycemic-glycogenolytic factor crystal powder.HPLC purity is more than 95%.
Produce hyperglycemic-glycogenolytic factor with above-mentioned technology, pancreas per ton can get the 0.3-0.4g hyperglycemic-glycogenolytic factor, HPLC purity is more than 95%, throw 1000 tons in pancreas per year and produce Regular Insulin calculating, can from discarded insulin sodium salt mother liquor, obtain the nearly 400g of hyperglycemic-glycogenolytic factor, by 30,000 yuan of every grams, be worth ten thousand yuan of 900-1200.And this technology is convenient to large-scale production.
Claims (4)
1. the preparation technology of a hyperglycemic-glycogenolytic factor, feature of the present invention are to be raw material with the waste insulin sodium salt crystalline mother solution of producing Regular Insulin.Mother liquor is transferred PH3-5 with 1-3N hydrochloric acid, with centrifugal or remove by filter the acidic protein of separating out, the zinc acetate or the zinc oxide that in filtrate, add 0.01-0.1M then, the PH5-7.5 precipitation is transferred with 1-3N sodium hydroxide in the back, to precipitate with dilute acetic acid or diluted hydrochloric acid dissolution Shangyang ion-exchange gel post, use the weak ammonia wash-out then, elutriant is added Zn again
++Secondary sedimentation, the zinc precipitation is dissolved with the saliniferous dilute acetic acid, last gel-filtration column, the hyperglycemic-glycogenolytic factor honeybee that gel-filtration column is washed out concentrates through cationic exchange coloum, then concentrated solution is transferred the PH7-9 crystallization with dilute phosphoric acid, use the phosphoric acid buffer systems recrystallization again, crystallization gets 12 body hyperglycemic-glycogenolytic factor crystal powders after collection, dehydration, drying, and its technical process is:
2. process for preparing glucagon according to claim 1 is characterized in that said cationic exchange gel column is an ion exchange column.
3. process for preparing glucagon according to claim 1, it is characterized in that also can be with insulin sodium salt crystalline mother solution thin up to 2-10 times of volume; With behind the 1-3N hydrochloric acid accent PH3-5 cationic exchange coloum on the solution being replaced a Zn
++Precipitation.
4. according to claim l and 3 described hyperglycemic-glycogenolytic factor preparation methods, it is characterized in that said ion exchange column is an ion-exchange expansion column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00112488 CN1340549A (en) | 2000-08-26 | 2000-08-26 | Process for preparing glucagon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00112488 CN1340549A (en) | 2000-08-26 | 2000-08-26 | Process for preparing glucagon |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1340549A true CN1340549A (en) | 2002-03-20 |
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Family Applications (1)
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| CN 00112488 Pending CN1340549A (en) | 2000-08-26 | 2000-08-26 | Process for preparing glucagon |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102940879A (en) * | 2003-06-03 | 2013-02-27 | 诺沃挪第克公司 | Stabilized pharmaceutical peptide compositions |
| CN103709244A (en) * | 2012-09-29 | 2014-04-09 | 宜昌长江药业有限公司 | Purification method for insulin crystal or insulin analogue crystal |
-
2000
- 2000-08-26 CN CN 00112488 patent/CN1340549A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102940879A (en) * | 2003-06-03 | 2013-02-27 | 诺沃挪第克公司 | Stabilized pharmaceutical peptide compositions |
| CN102940879B (en) * | 2003-06-03 | 2017-06-06 | 诺沃挪第克公司 | Stabilized pharmaceutical peptide compositions |
| CN103709244A (en) * | 2012-09-29 | 2014-04-09 | 宜昌长江药业有限公司 | Purification method for insulin crystal or insulin analogue crystal |
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